HGF (hepatocyte growth factor) is a multifunctional heterodimeric polypeptide produced by mesenchymal cells. HGF is composed of an alpha-chain containing an N-terminal domain and four kringle domains (NK4) covalently linked to a serine protease-like beta-chain C-terminal domain (see FIG. 1). Human HGF is synthesized as a biologically inactive single chain precursor consisting of 728 amino acids with a 29 amino acid signal peptide which is not present in the mature protein. Biologically active HGF is achieved through cleavage at the R494 residue by a specific, extracellular serum serine protease. The active HGF thus achieved is a fully active heterodimer which is composed of disulfide linked 69 kDa alpha-chain and 34 kDa beta-chain. However, the overall tertiary structure of HGF is still unknown and it has not yet been clarified which of these domains is responsible for the specific functions of HGF (Maulik et al., Cytokine & Growth Factor Reviews 13(1): 1-59, 2002).
The binding of HGF to its receptor, Met, induces the growth and scattering of various cell types, mediates the epithelial mesenchymal transitions and the formation of tubules and lumens, and promotes angiogenesis. Both Met and HGF knockout mice are embryonic lethal and show developmental defects in placenta, fetal liver and limb/muscle formation (Cao et al., PNAS 98(13): 7443-7448, 2001; Gmyrek et al., American Journal of Pathology 159(2): 579-590, 2001).
Met was originally isolated as a product of a human oncogene, trp-met, which encodes a constitutively active altered protein kinase with transforming activity. Met activation has also been shown to remarkably enhance the metastastic spread of cancer stemming from its stimulatory influence of processes such as angiogenesis, cell motility, and cell surface protease regulation (Wielenga et al., American Journal of Pathology 157(5): 1563-1573, 2000). Since Met was reported to be over-expressed in various human cancers of liver, prostate, colon, breast, brain and skin (Maulik et al, supra), it has been regarded as an important target factor for the prevention and treatment of cancer. Further, it has been reported that malaria infection depends on activation of the HGF receptor by secreted HGF, and accordingly, HGF and its receptor are identified as potential targets for new approaches to malaria prevention (Carrolo M, et al., Nat. Med. 9(11): 1363-1369, 2003). It has been also discovered the possibility that HGF may be found in association with the pathologic changes which occur in Alzheimer's disease (Fenton H, et al., Brain Res. 779(1-2): 262-270, 1998). Furthermore, it has been found that HGF is definitely involved in enhancing cutaneous wound healing processes, including re-epithelialization, neovascularization and granulation tissue formation (Yoshida S, et al., J. Invest. Dermatol. 120(2): 335-343, 2003).
Meanwhile, selective neutralization of tumor-associated growth factors or cytokines and their receptors, which play crucial roles in the development and spread of cancer, has always been an attractive strategy for the development of anti-cancer drugs. Recently, numerous therapeutic monoclonal antibodies (mAbs) for these targets, e.g., herceptin, and anti-angiopoietin human mAbs have been developed using recombinant antibody technologies such as phage display of combinational antibody library.
It is well known that polyclonal antibodies against HGF block many of HGF biological functions. In addition, it has been recently reported that mixtures of neutralizing mAbs against HGF display anti-tumor activity in animal models (Cao et al., PNAS 198(13): 7443-7448, 2001). In particular, Cao et al. disclosed that three or more of the epitopes, possibly two for the Met receptor and one for heparin, need to be blocked in order to inhibit HGF activity in vivo and in vitro, and a mixture of at least 3 mAbs is capable of neutralizing HGF in an in vitro experiment.
However, there has been reported no monoclonal antibody that can neutralize HGF as a single agent and inhibit cell scattering activity in vitro.